Field of the Invention
The present invention relates generally to the field of diagnostic methods and biomarkers relating to myasthenia gravis. More specifically, the present invention relates to muscle specific tyrosine kinase-fluorophore conjugates compositions, kits and methods of using the compositions.
Description of the Related Art
Myasthenia gravis is a neuromuscular disorder characterized by weakness and fatigability of skeletal muscles. The underlying defect is a decrease in the number of available acetylcholine receptors at neuromuscular junctions due to an antibody-mediated autoimmune attack. In the neuromuscular junction, acetylcholine is synthesized in the motor nerve terminal and stored in vesicles (quanta). When an action potential travels down a motor nerve and reaches the nerve terminal, acetylcholine from 150 to 200 vesicles is released and combines with acetylcholine receptors that are densely packed at the peaks of postsynaptic folds. When acetylcholine combines with the binding sites on the acetylcholine receptor, the channels in the acetylcholine receptors open, permitting the rapid entry of cations, chiefly sodium, which produces depolarization at the end-plate region of the muscle fiber. If the depolarization is sufficiently large, it initiates an action potential that is propagated along the muscle fiber, triggering muscle contraction. This process is rapidly terminated by hydrolysis of acetylcholine by acetylcholinesterase and by diffusion of acetylcholine away from the receptor.
In myasthenia gravis, the fundamental defect is a decrease in the number of available acetylcholine receptors at the postsynaptic muscle membrane. In addition, the postsynaptic folds are flattened, or “simplified.” These changes result in decreased efficiency of neuromuscular transmission. Therefore, although acetylcholine is released normally, it produces small end-plate potentials that may fail to trigger muscle action potentials. Failure of transmission at many neuromuscular junctions results in weakness of muscle contraction.
The neuromuscular abnormalities in myasthenia gravis are brought about by an autoimmune response mediated by specific anti-acetylcholine receptor antibodies. The anti-acetylcholine receptor antibodies are called pathogenic antibodies and reduce the number of available acetylcholine receptors at neuromuscular junctions by three distinct mechanisms: (1) accelerated turnover of acetylcholine receptors by a mechanism involving cross-linking and rapid endocytosis of the receptors; (2) blockade of the active site of the acetylcholine receptor, i.e., the site that normally binds acetylcholine; and (3) damage to the postsynaptic muscle membrane by the antibody in collaboration with complement. The pathogenic antibodies are IgG and are T-cell dependent.
The clinical manifestations of the autoimmune disease myasthenia gravis are correlated with the presence of these pathogenic antibodies located at the neuromuscular junction. Up to date, only a few therapies exist, which are either symptomatic treatment or immunotherapy.
Seventy percent of patients with myasthenia gravis carry autoantibodies to the acetylcholine receptor and a separate 5-10% carry autoantibodies to muscle specific tyrosine kinase (Vincent and Leite, 2005, Curr Opin Neurol; 18(5):519-25). Symptoms of patients with muscle specific tyrosine kinase related myasthenia gravis include fatigue, muscle weakness, double vision, drooping eyelids, and difficulty chewing or swallowing and in severe disease paralysis and respiratory distress. Muscle specific tyrosine kinase antibodies are mainly of the non-complement-fixing IgG4 isotype. Muscle specific tyrosine kinase-myasthenia gravis is most commonly diagnosed by RIA/ELISA. Electrophysiological studies, such as single-fiber electromyography, are often used to diagnose acetylcholine receptor-seronegative myasthenia gravis. However, these studies are time-consuming, require specialized expertise, have limited availability, and are unpleasant for patients. The limitations of this assay are a prolonged turnaround time and few labs offering the test. Serum muscle specific tyrosine kinase antibody concentrations are also not a reliable marker for disease activity or severity.
There is a recognized need, therefore, for muscle specific tyrosine kinase-fluorophore conjugates compositions and kits and methods of using the compositions in the diagnosis of myasthenia gravis. The present invention fulfills this longstanding need and desire in the art.